Application of riluzole in the treatment of mitochondrial diseases

ABSTRACT

A method for the treatment of mitochondrial diseases, including Kearns-Sayre syndrome, MERRF syndrome, MELAS syndrome and Leber&#39;s disease, is carried out by administering to a patient in need of treatment an effective amount of riluzole or a pharmaceutically acceptable salt thereof.

This application is a 371 of PCT/FR95/00023 Jan. 9, 1995.

The present invention relates to a novel therapeutic application ofriluzole or the pharmaceutically acceptable salts of this compound.

Riluzole, or 2-amino-6-trifluoromethoxybenzothiazole, is useful as ananticonvulsant, anxiolytic and hypnotic medicinal product (EP Patent 50551), in the treatment of schizophrenia (EP 305 276), in the treatmentof sleep disorders and depression (EP 305 277), in the treatment ofcerebrovascular disorders and as an anaesthetic (EP 282 971).

It has now been found, surprisingly, that this compound may also be usedin the treatment of mitochondrial diseases.

Mitochondrial diseases are degenerative diseases which are linked todifferent mechanisms such as, for example, anomalies of mitochondrialDNA (deletions, point mutations, depletions, duplications), anomalies ofcellular DNA coding for mitichondrial enzymes or complex macromolecularmitochondrial elements, acquired conditions caused by toxic substances(for example MPTP or CO) or by medicinal products (for examplechloramphenicol, AZT or acetylsalicylic acid). Mitochondrial diseaseswhich may be mentioned are Kearns-Sayre syndrome, MERRF syndrome(Myoclonic Epilepsy with Ragged Red Fibres), MELAS syndrome(Mitochondrial Encephalopathy, Myopathy, Lactic Acidosis and Stroke-likeepisodes) and Leber's disease (I. Nonaka, Current Opinion in Neurologyand Neurosurgery, 5 (1992) 622).

The action of riluzole in the treatment of mitochondrial diseases isdemonstrated by its protective effect in the cyanide test. It is knownin fact that potassium cyanide exerts a toxic effect by inhibition ofcytochrome oxidase aa3, the terminal enzyme in the electron transportsequence at the mitochondrial level. Thus in mice potassium cyanide (3mg/kg i.v.) induces abdominal contractions and tonic attacks followed bydeath within 20 seconds.

Male adult mice weighing 22-25 g (CD1 COBS, Charles River), housed undercontrolled temperature and lighting conditions, receive food anddrinking water ad libitum. The vehicle or the product to be studied isgiven to groups of 6 animals, either intraperitoneally 30 minutes beforethe intravenous bolus administration of a lethal dose (3 mg/kg) ofpotassium cyanide, or orally 1 hour before the administration ofpotassium cyanide. In the control group, all of the mice die within 20seconds, whereas the ED50 for riluzole i.p. is 4.5 mg/kg and the ED50for riluzole p.o. is 7.8 mg/kg.

The action of riluzole in the treatment of mitochondrial diseases isalso demonstrated by its effect on the mitochondrial metabolism of therat liver.

The metabolic activity of the mitochondrion is monitored by theconsumption of oxygen by polarography, using a Clark electrode (marketedby Bioblock). The experimental setup consists of a glass chamberthermostated at 27° C. by a hot water system. A magnetic bar placedwithin the chamber permits continuous stirring of the reaction mediumand facilitates the establishment of equilibrium between the dissolvedoxygen and the gas diffusing through the membrane of the electrode. Themitochondria, the substrate (succinate) and ADP (adenosine diphosphate)are introduced into the reaction chamber through a small aperturesituated in the upper part of the apparatus. A Clark electrode isimmersed in the reaction medium via an opening which is hermeticallysealed by a screw stopper. The consumption of oxygen is plotted by arecorder which is connected to a YSI model 5300 Biological OxygenMonitor (marketed by Bioblock).

Mitochondria from the liver of the male SD rat are isolated at +4° C. bydifferential centrifugation according to the method described byAppelmans et al., Biochem. J., 59 (1955) 438-445. The mitochondrialpellet is taken up in from 1 to 3 ml of 0.25M sucrose buffer andhomogenized using a pipette.

The oxygen consumption of the mitochondria (1.2 mg of proteins per ml)is measured in 5 ml of Chance buffer, consisting of 12 mM of NaF, 26 mMof NaCl, 58 mM of KCl and 3 mM of succinate substrate, so as to bringabout the metabolic state 4 of Chance-Williams (or mitochondrial redoxstate 4, Chance B., Energy liked functions of mitochondria, New York,Acad. Press 1963)). The temperature of 27° C. corresponds to an oxygenconcentration of approximately 250 μM. The metabolic state 3 ofChance-Williams is brought about by addition of 0.25 mM of adenosinediphosphate in control preparations containing 250 nM of riluzole.

The mean oxygen consumption of the control hepatic mitochondria is 1.63μmol/min/mg of proteins (3 tests). In the presence of 250 nM of riluzoleadded in vitro to the mitochondria, the mean oxygen consumption is 2.12μmol/min/mg of proteins (3 tests). These results show that riluzolestimulates mitochondrial respiration by about 30% relative to thecontrols.

Pharmaceutically acceptable salts which may be mentioned in particularare the addition salts with inorganic acids, such as hydrochloride,sulphate, nitrate or phosphate, or with organic acids, such as acetate,propionate, succinate, oxalate, benzoate, fumarate, maleate,methanesulphonate, isethionate, theophyllineacetate, salicylate,phenolphthalinate or methylenebis(β-hydroxynaphthoate), or substitutionderivatives of these derivatives.

The medicinal products consist at least of riluzole, in free form or inthe form of an addition salt with a pharmaceutically acceptable acid, inthe pure state or in the form of a composition in which it is combinedwith any other pharmaceutically compatible product, which may be inertor physiologically active. The medicinal products according to theinvention may be employed orally, parenterally, rectally or topically.

Solid compositions for oral administration which may be used aretablets, pills, powders (gelatin capsules, wafer capsules) or granules.In these compositions the active principle according to the invention ismixed with one or more inert diluents such as starch, cellulose,sucrose, lactose or silica, under a stream of argon. These compositionsmay also comprise substances other than diluents, for example one ormore lubricants such as magnesium stearate or talc, a colouring, acoating (coated tablets) or a varnish.

Liquid compositions which may be used for oral administration arepharmaceutically acceptable solutions, suspensions, emulsions, syrupsand elixirs, containing inert diluents such as water, ethanol, glycerol,vegetable oils or liquid paraffin. These compositions may comprisesubstances other than diluents, for example wetting, sweetening,thickening, flavouring or stabilizing products.

The sterile compositions for parenteral administration may preferably besolutions, aqueous or non-aqueous, suspensions or emulsions. As asolvent or vehicle, water, propylene glycol, polyethylene glycol,vegetable oils, especially olive oil, injectable organic esters, forexample ethyl oleate, or other suitable organic solvents may beemployed. These compositions can also contain adjuvants, especiallywetting, isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization may be carried out in several ways, for example by asepticfiltration, by incorporation of sterilizing agents in the composition,by irradiation or by heating. They may also be prepared in the form ofsterile solid compositions which can be dissolved at the time of use insterile water or any other sterile injectable medium.

The compositions for rectal administration are suppositories or rectalcapsules which contain both the active product and excipients such ascocoa butter, semisynthetic glycerides or polyethylene glycols.

The compositions for topical administration may for example be creams,lotions, mouth washes, nasal drops or aerosols.

The doses depend on the desired effect, on the duration of treatment andon the administration route used; they are generally between 50 and 400mg per day by the oral route for an adult, with single doses rangingfrom 25 to 200 mg of active substance.

Generally speaking, the doctor will determine the appropriate dosage inaccordance with the age, the weight and all of the other factorsspecific to the subject to be treated.

The examples which follow illustrate medicinal products according to theinvention:

EXAMPLE A

The usual technique is used to prepare tablets containing a 50 mg doseof active product and having the following composition:

2-Amino-6-trifluoromethoxybenzothiazole 50 mg

Mannitol 64 mg

Microcrystalline cellulose 50 mg

Polyvidone excipient 12 mg

Sodium carboxymethylstarch 16 mg

Talc 4 mg

Magnesium stearate 2 mg

Anhydrous colloidal silica 2 mg

Mixture of methylhydroxypropylcellulose, polyethylene glycol 6000 andtitanium dioxide (72:3.5:24.5)

q.s. 1 finished film-coated tablet weighing 245 mg

EXAMPLE B

The usual technique is used to prepare hard gelatin capsules containinga 50 mg dose of active product and having the following composition:

2-Amino-6-trifluoromethoxybenzothiazole 50 mg

Cellulose 18 mg

Lactose 55 mg

Colloidal silica 1 mg

Sodium carboxymethylstarch 10 mg

Talc 10 mg

Magnesium stearate 1 mg

EXAMPLE C

An injectable solution is prepared which contains 10 mg of activeproduct and has the following composition:

2-Amino-6-trifluoromethoxybenzothiazole 10 mg

Benzoic acid 80 mg

Benzyl alcohol 0.06 cm3

Sodium benzoate 80 mg

Ethanol, 95% 0.4 cm3

Sodium hydroxide 24 mg

Propylene glycol 1.6 cm3

Water q.s. 4 cm3

The invention also relates to the process for the preparation ofmedicinal products which can be used in the treatment of mitochondrialdiseases, consisting in mixing riluzole or the pharmaceuticallyacceptable salts of this compound with one or more compatible andpharmaceutically acceptable diluents and/or adjuvants.

We claim:
 1. A method for the treatment of at least one mitochondrialdisease, said method comprising administering to a patient in recognizedneed of said treatment an amount of riluzole or a pharmaceuticallyacceptable salt thereof effective for the treatment of said at least onemitochondrial disease.
 2. The method of claim 1, wherein saidmitochondrial disease is Kearns-Sayre syndrome, MERRF syndrome, MELASsyndrome or Leber's disease.
 3. A method according to claim 1, whereinsaid riluzole or a pharmaceutically acceptable salt thereof isadministered in an amount ranging from 50 to 400 mg per day.
 4. A methodaccording to claim 1, wherein said riluzole or a pharmaceuticallyacceptable salt thereof is administered in a dose ranging from 25 to 200mg of active substance.